The sustainable production of plant bioactive compounds is increasingly important as natural habitats decline. This study investigates whether in vitro cell cultures of
Eryngium planum,
Lychnis flos-cuculi, and
Kickxia elatine can serve as alternative sources of biologically active biomass with antimicrobial and anti-
Acanthamoeba properties. Callus cultures were established under optimized and controlled conditions, and metabolomic profiling was completed using UPLC-HRMS/MS. In silico analysis, using a molecular docking approach, was applied to understand the interaction between target compounds and
Acanthamoeba profilin and identify possible targets for antimicrobial properties. Untargeted metabolomic analysis confirmed the presence of valuable compounds in the callus cultures of the studied species. Biological activity was assessed through anti-
Acanthamoeba and antimicrobial assays.
Lychnis flos-cuculi and
Kickxia elatine callus extracts showed significant inhibitory effects on
Acanthamoeba trophozoites, with 87.5% and 80.1% inhibition at 10 mg/mL, respectively. In contrast,
E. planum extract stimulated amoebic growth. The anti-
Acanthamoeba activity correlated with the presence of ferulic acid and
p-coumaric acid in
L. flos-cuculi extract, and acteoside in
K. elatine extract. Antibacterial testing revealed moderate activity of
E. planum and
K. elatine extracts against
Staphylococcus spp., while Gram-negative bacteria and fungi were largely resistant. These findings highlight the potential of in vitro cultures—particularly those from
L. flos-cuculi and
K. elatine—as promising, sustainable sources of anti-
Acanthamoeba and antimicrobial agents, warranting further investigation into their pharmacologically active constituents.
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